Abstract The incidence of human papillomavirus associated (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) is rising in the US, and is now even more prevalent than cervical cancer. Due to low HPV vaccination rates and the decades long latency period between HPV infection and cancer diagnosis, HPV+ OPSCC remains a major health concern, with the most likely reason for death being distant metastasis. Furthermore, due to the life-long detrimental treatment effects on quality of life for survivors, it is essential to identify a subset of patients who would benefit from de-escalated treatment. Our long term goal is to differentiate HPV+ patients who have a good prognosis and are most likely to benefit from de-escalated therapy and those who require the standard, or a more aggressive regimen. Our group first characterized two main subtypes of HPV+ OPSCC, identifying HPV integration into the host genome as the driving factor in determining tumor subtype. Furthermore, we and others have shown that HPV integration status is associated with overall survival. In this proposal we plan to study three downstream effects of HPV integration identified by our group and others: an increase in the splicing of HPV oncogene E6 to E6*, a decrease in the tumor immune response, and a change in cell differentiation status. Each of these effects plays a role in determining metastasis and survival, however the mechanism of their effect and their relative contributions remain unclear. In aim 1, we will disentangle the above three effects of HPV integration on overall and disease-specific survival using a University of Michigan cohort of 300 patients, and we will compare methods for defining HPV integration status. We will also optimize a biomarker for tumor immune infiltration based on H&E slides. In aim 2, we will examine the oncogenic effects of the shift to expressing the shorter E6* isoform instead of full length E6, using in vitro and in vivo models. This was observed by us and others to increase oxidative phosphorylation and potentially tumor mutational burden. Finally, in aim 3 we will use in vitro and vivo models to examine mechanism by which HPV integration and/or the shift to E6* expression modulate cell invasion and treatment response. Based on our results, we will begin one or more biomarker-based pilot phase II clinical trials.